The separation of macromolecules from biological samples by electrophoretic techniques has been a relatively common practice for at least twenty years, and many different devices and techniques have been developed to accomplish various desirable ends. Early techniques involed imposing an electric field across a slab of gel and placing a sample of material to be analyzed on one end of the gel. Macromolecules exhibit varying mobility in a properly prepared gel depending on a number of variables, such as an electric charge, size and relative mass of the different species, and shape of the macromolecular species, which may be influenced by a strong electric field. Due to these variations, different species will move into and through a gel at differing rates, forming distinct bands as they move through the gel, thus accomplishing separation. The separate bands are sometimea called fractions, as they are each a portion of the original sample. After separation is accomplished in a gel, the electric field may be discontinued and the gel removed from any support that is used. There are techniques for rendering the fractions identifiable, such as staining and radioactive tagging, so a spectrum may be recorded. By comparing such spectra with empirical spectra produced from known mixtures and concentrations of such materials, the particular material of each fraction from an experimental sample may be identified. Techniques have also been developed for continuous elution of bands of separated fractions as they move off the end of a gel column.
Gel apparatus may take many different forms in the art, and, in the various different designs, the most common geometries for the gel region are gel slabs and gel columns. These structures are usually prepared by first mixing chemicals, including one or more reacting agents that promote curing of some of the liquid material into a gelled state. A support structure, such as a tube, or two spaced apart flat plates, is then filled with the mixture, and reactions take place to form the gel in the support structure. Typically a starting material is a monomer, an initiator, and one or more of several crosslinking agents, and surfactants. An aqueous buffer is typically included to provide an electrically conductive medium in the gel, compatible with buffers that may be used outside the gel in an electrophoresis system. Other chemicals, such as Urea as a denaturing agent, may be included as well. Gels may be of different composition as well, two common compositions being polyacrylamide and agarose, although agarose gels are, in the strict sense, not polymer gels.
In gel electrophoresis, the gel is commonly cast in an aqueous solution including an ionic buffer, and an electrical potential is applied across the gel. The electrical potential is responsible for the force causing molecules to migrate through the gel, also induces an electric current. The passage of the electric current in traditional systems, with gel slabs and columns having thickness and diameters of several millimeters and greater, has been a problem in many instances due to Joule heating. Such heating, for example, can cause distortion of the gel structure and subsequent interference with the separation process. To overcome the Joule heating effects, electrophoresis apparatus is often complicated and bulky, including elaborate elements and structures for removing heat.
The heating problem has led in the art to construction of apparatus with smaller and smaller gel structures. At the present time, the industry is headed toward the use of very thin slabs and rectangular and cylidrical capillaries filled with gel. In principle, the thin-wall and small diameter structures should prove very effective as the surface area of the supporting structures relative to the bulk of the gel is larger than in traditional structures. Although the heat per unit volume generated would be the same, the heat transfer away from the gel should be facilitated. In these thin structures, the preferred thickness of slabs would be in the range of from tens to hundreds of microns. A number of recent publications discuss the relative merits of capillaries of narrow dimensions for gel electrophoresis, for example, see A. S. Cohen and B. L. Karger, J. Chromatography, 397, 409 (1987); and S. Hjerten, et al., J. Chromatography, 403, 47 (1987).
The usefulness of a cast gel for biomolecular separation procedures depends upon a number of variables. The relative degree of crosslinking is important to the migration of macromolecules, for instance, and the homogeneity of the gel may be important. In many cases, the gel must be firmly adhered to the walls of the support structure, typically glass or plastic, so that the gel material does not migrate in the system due to electroendosmosis. Some work has been performed in this area as reported in Hjerten et al. (ibid ), which described the importance of wall treatment in suppressing adsorption of sample solutes onto the walls. Such coatings included for example, methylcellulose or linear polyacrylamide.
The gel must be continuous, too. The appearance of voids, particularly with thin slabs of capillary dimensions, can render a gel structure useless. A void can cause an anomaly in the continuity of the electrical circuit, or may seriously alter the nature of macromolecular bands as they migrate. The appearance of such voids has been a particularly vexatious problem in the preparation of such gels in systems of capillary dimensions, despite the fact that extensive fundamental research concerning the polymerization kinetics and product gel behavior has been reported in the literature. (see, for example, A. Chrambach and C. Rodbard, Separation Sciene, 7, 213 (1981; ) C. Gelfi and PlG. Righetti Electrophoresis, 2, 213 and 220 (1981); and P. G. Righetti, et al. Electrophoresis, 2, 291 (1981).)
In the casting of slab gel structures, there are applications for which it is desirable that the composition of gel vary along one direction. For example, by having a varying "pore" size, a wider range of sizes of molecules can be better separated. The resulting gel structure is called a gradient gel. At the present time, such slabs with a gradient in compostition are made using a mixing pump to premix the monomer/crosslinker/buffer solution continuously in order to vary the mixture composition being poured onto an open-face support. This is typically accomplished by preparing a liquid gel mixture with a first set of concentrations of polymerizing materials in one reservoir, then pouring from the first reservoir onto the support structure while adding materials with a second set of concentrations of polymerizing materials to the first reservoir, so that the combined materials poured onto the support structure are successively altered. The resulting liquid mixture on the support structure is then relatively quickly polymerized (gelled) so that natural diffusion doesn't obliterate the gradient to any significant degree before gellation takes place. Then a top-face is placed over the gel. This technique is adequate for slabs of large dimension, e.g. having channels of the order of a millimeter in thickness. This process is difficult to manage and control even for open structures, and is all but impossible for narrow channels, closed slabs, where the crosslinked gel is to bind with the glass surfaces.
All of these problems need to be overcome in the casting of gel structures. What is needed is an apparatus and method for preparing gel structures that is reliable, in which the degree of crosslinking can be controlled, that results in structures without voids, and by which the polymer concentration can be varied from one end of the slab to the other.